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Notes: These authors investigated the effects of various anesthetics on bioluminescence imaging with firefly luciferase. They observed decreases in luminescence with volatile anaethetics, and found increased luciferase expression with injectable anaethetics in intact cells, but not in cell lysates in vitro. They concluded that the decreases in luciferase activity observed with volatile anaesthesia were due to hemodynamic effects, and not due to a direct inhibitory effect on luciferase enzyme itself. The apparent enhancement of luciferase activity with certain injectable anaesthetics appeared to be due to cytotoxic effects that resulted in increased permeablity to luciferase, as the same enhancement was not observed in cell lysates. D-Luciferin was used for in vivo imaging experiments. The pGL4.10 vector (encoding firefly luciferase), Luciferase Assay Reagent, and the GloMax® 96 Microplate Luminometer were used for in vitro assays using cell lysates. (4190)

Notes: These authors used the Flexi® Vector System to prepare ORF clones encoding 1929 human genes and to transfer a subset of these clones to various expression vectors for further analysis. They created HaloTag® fusion proteins and examined expression of these proteins in vitro and in COS7 and HEK293 cells. They also performed comparisons between the Flexi® System and Gateway® cloning system, specifically examining the effects of flanking sequences on protein expression in in vitro translation systems and confirming that the cellular localization of the HaloTag® fusion proteins was consistent with results obtained using GFP-fusions. (3800)

Notes: Oxidative damage has been associated with a range of age-related neurological conditions. In this study, the effect of mRNA oxidation was investigated. A direct correlation was observed between the extent of oxidation and the frequency of translation errors. The authors excised the firefly luciferase (luc2) gene from the pGL4.14 Vector, attached a FLAG tag to the 5´ terminus and a Myc tag to the 3´ terminus, and subcloned the gene into a pGEM-4Z Vector that had been modified to append a poly(A) sequence. The construct was transfected into HEK293 cells, which were then cultured in the presence of an oxidizing agent. The occurrence of truncated protein fragments and short peptides increased in the presence of the oxidizing agent in a concentration-dependent manner. The effects of oxidation of mRNA were also investigated in in vitro translation experiments using mRNA treated with an iron-ascorbate mixture and hydrogen peroxide. Translation in vitro was performed using rabbit reticulocyte lysate supplemented with protease inhibitors. The translation products were detected using anti-FLAG and anti-c-Myc antibodies. (3630)

Notes: In this family-based study of 1,231 autism cases, a genetic association of a common C allele in the promoter of the MET gene and autism was identified. Initial sequencing of the MET genes from 86 individuals with autism was used to identify several candidate variants in the MET promoter and 3´ untranslated region. Family-based analyses were then performed to determine whether an association could be demonstrated between any of these variants and autism. A G/C variant 20bp 5´ of the MET transcription start site was found to be overrepresented among individuals with autism, particularly among families where more than on child was affected. Once the candidate variant was identified, the effect of the G/C change on transcription of the MET gene was investigated in a reporter assay. Two 762 bp fragments of the MET promoter region, differing only in the G/C nucleotide, were cloned into pGL4.10 firefly luciferase reporter vectors. These vectors were then transfected into mouse neural cell lines, and luciferase production was monitored using the Dual-Glo™ Assay. The construct containing the C allele produced less than half of the luciferase activity of construct containing the G allele. (3579)

Cancer Res.66, 11613-11622.
HMGA1 is a determinant of cellular invasiveness and in vivo metastatic potential in pancreatic adenocarcinoma.2006

Liau, S-S, Jazag, A. and Whang, E.

Notes: The HMGA1 proteins are known to play an important role in transcriptional regulation of human gene expression, and are overexpressed in several malignancies. This study investigated the role of HMGA1 in cellular invasiveness and metastasis of pancreatic adenocarcinoma. Overexpression of HMGA1 in the human pancreatic cell line MiaPaCa2 resulted in increased invasiveness in a Matrigel assay, and shRNA silencing of HMGA1 expression resulted in reduced invasiveness and reduced the activity of matrix metalloproteinase-9 (MMP-9), an important mediator of malignant cell invasiveness. The authors then used a reporter assay to investigate the effect of changes in HMGA1 expression on MMP-9 activity. Vectors either overexpressing HMGA1, expressing an shRNA directed against HMGA1, or expressing a control shRNA, were cotransfected into MiaPaCa2 cells along with a pGL4.12 firefly luciferase reporter vector construct containing the MMP-9 promoter upstream of the luciferase gene. MMP-9 promoter activity was significantly decreased upon HMGA1 silencing. The authors then showed that HMGA1 silencing resulted in decreased invasiveness and reduced tumor growth and MMP-9 activity in an in vivo model. (3578)

Notes: These authors used stable-isotope labeling of amino acids in culture (SILAC), a method that allows quantitation of relative protein abundance between populations, to investigate the effect of the microRNA miRNA-1 on the HeLa cell proteome. HeLa cells grown in the presence of labeled arginine and lysine were transfected with miRNA-1, and the labeled proteins compared to those from control cells treated with the transfection reagent alone. A set of 16 proteins repressed by miRNA-1 was identified. The 3´ UTR's from 11 of the miRNA-1-regulated genes were tested in a reporter assay, and 6 were shown to repress expression in an miRNA-1-dependent fashion. For the reporter assays, the HSV TK promoter was amplified from the pRL-TK Vector and subcloned into the pGL4.12(luc2CP) Vector, creating the pGL4.12-TK vector. The 3´ UTR regions from suspected target genes were then amplified and subcloned into the pGL4.12-TK construct. The various pGL4.12-TK constructs (0.9µg) were then co-transfected along with pRL-TK (0.1µg) and miRNA-1 (50pmol) into HeLa cells (80,000 cells/well in 12-well plates). Twenty-two hours post-transfection, firefly and Renilla luciferase activities were measured using the Dual-Luciferase® Reporter Assay System. (3561)

Notes: Three alleles were identified in the promoter of the serotonin transporter, HTTLPR, based on the number of a specific repeat sequence and a single nucleotide polymorphism found in the repeat sequence. Sequences from these alleles (named S, LA, and LG) were cloned into the pGL4.10 [luc2] Vector. These constructs were then tested by transfecting them into RN46A cells derived from rat dorsal raphe neurons. The constructs containing the LG and S allele sequences displayed a 2.8 fold increase in activity compared to the construct containing the LA allele sequence. Data was represented as relative luciferase levels compared to a control (pGL4.10 [luc2] Vector). (3411)

Notes: The transcription factors BMAL1 and CLOCK form a heterodimer that positively regulates the Period (Per) and Cryptochrome (Cry) genes, which are involved in maintenance of circadian oscillations in mammals. The PER and CRY proteins negatively regulate the activity of the BMAL1/CLOCK heterodimer. To investigate the specific role of BMAL1 in maintenance of circadian rhythms, Per or Bmal1 promoter-driven luciferase reporter vectors were created using the pGL4.11[luc2P] luciferase reporter vector. Forty-eight hours after transfection of Rat-1 fibroblasts with each reporter construct, cellular clocks were synchronized by treatment with 100nM dexamethasone, and rhythmic expression of the luciferase reporter was observed. The effect of various mutant BMAL1 constructs on luciferase expression was then evaluated using this model system. (3472)

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